Glycosaminoglycans (GAG)s modulate enzyme activities (e.g., of antithrombin III or heparin cofactor II), regulate cell behaviors (e.g., cell adhesion, growth, and differentiation), and control the function of extracellular matrices (e.g., diffusion of ions through basement membranes, and fibrillogenesis and lateral associations of collagens), largely through non-covalent interactions with proteins. (Jackson, R. L., et al., Physiol Rev 71:481-539, 1991; Lindahl, U. and M. Hook, Ann Rev Biochem 47:385-417, 1978). Although many proteins exhibit high affinity interactions with heparan sulfate or heparin and other GAGs, the specificity of such interactions has been defined for only a small number of them. (San Antonio, J. D. and R. V. Iozzo, Encycl Life Sci In Press, 2000). As heparan sulfates and heparin are among the most structurally diverse and biologically active of GAGs, their protein-interactive features have been the most thoroughly studied. Fine structural features of heparan sulfate chains, including defined sequences, rare modifications, domain structures, and gross polymer characteristics are each believed to contribute to various classes of interactions with different proteins. (San Antonio, J. D., and R. V. Iozzo, Encycl Life Sci In Press, 2000). For proteins, domains rich in basic amino acids appear to be necessary to facilitate interactions with GAGs; and for a subset of these proteins, potential heparin-binding consensus sequences have been described. (Cardin, A. D. and H. J. R. Weintraub, Arteriosclerosis, 9:21-32, 1989; Jackson, R. L., et al., Physiol Rev 71:481-539 1991).
Heparin-binding consensus sequences were discovered by Cardin and Weintraub, who surveyed amino acid sequences of known heparin-binding proteins, where they identified two potential consensus sequence motifs for heparin-binding, X-B-B-X-B-X or X-B-B-B-X-X-B-X, where X represents a hydropathic or uncharged amino acid, and B a basic amino acid. (Cardin, A. D. and H. J. R. Weintraub, Arteriosclerosis 9:21-32, 1989). For example, such consensus sequences were identified in proteins including apolipoprotein B-100, apo E, and vitronectin, to name a few. (See Cardin and Weintraub, 1989, for review). Molecular modeling of these consensus sites predicts the arrangement of amino acids into either α-helices or β-strands. This allows for the clustering of noncontiguous basic amino acids on one side of the helix, thus forming a charged domain to which GAGs could bind. Indeed, for some heparin-binding proteins, disruption of the heparin-binding consensus sequences hinders heparin binding. For example, chemical modification of the heparin-binding consensus site in thrombospondin (Lawler, J. and R. O. Hynes, Cell Biol 103:1635-1648, 1986) or site-directed mutagenesis of a heparin-binding sequence in fibronectin (FN) (Barkalow, F. J. B. and J. E. Schwarzbauer, J Biol Chem 266:7812-7818, 1991) eliminates or diminishes heparin-binding affinity. On the other hand, peptide mimetics of proposed heparin binding consensus sequences often fail to reveal the high affinities demonstrated by the native heparin-binding proteins. (Conrad, H. E, Heparin-Binding Proteins. Academic Press, 1998). Proteins often contain multiple, distal heparin-binding sequences that may come into proximity upon protein folding or multimerization, hence enabling binding through cooperativity. It has thus been speculated that the three dimensional arrangement of multiple heparin-binding consensus sites within or between heparin-binding proteins, and/or the presence of novel heparin-binding sites may be responsible for high affinity heparin- or HS-interactions with native proteins. Others have proposed a necessary approximately 20 Å distance between basic amino acids for heparin binding, regardless of protein tertiary structure. (Margalit, H., et al., J Biol Chem 268:19228-19231, 1993). Other heparin-binding sequences have been proposed that are variations of those reported by Cardin and Weintraub. The sequence TXXBXXTBXXXTBB, where T is a turn, was identified as a heparin-binding sequence in acidic FGF and bFGF. (Hileman, R. E., et al., BioEssays 20:156-167, 1998). X-ray crystallography revealed that this peptide backbone loops back upon itself in three turns to form a positively charged triangular heparin-binding pocket. The heparin-binding domain of von Willebrand factor resembles the motif XBBXXBBBXXBBX, a palindromic sequence in which the spacing and clustering of basic residues is important for heparin binding. (Sobel, M., et al., J Biol Chem 267:8857-8862, 1992). A third novel sequence has been demonstrated to be sufficient for weak heparin-binding in thrombospondin: WSXW. (Guo, N. H., et al., J Biol Chem, 267:19349-19355, 1992). However, for high affinity binding, this sequence must be flanked by basic residues. Other proteins including type I collagen (Sweeney, S. M., et al., PNAS 95:7275-7280, 1998), type VI collagen (Specks, U., et al., EMBO J,:4281-4290, 1992), extracellular-superoxide dismutase (Sandstrom, J., et al., J Biol Chem, 267:18205-18209, 1992), and mast cell chymases (Matsumoto, R., et al., J Biol Chem, 270:19524-19531, 1995), bind heparin via highly-basic binding regions which do not conform to any consensus sequence. In fact, in certain proteins, domains rich in basic amino acids have sometimes been shown to be unimportant for heparin binding. For example, the two heparin-binding consensus sequences identified in the FGFs were shown not to mediate heparin-binding (Wong, P., et al., J Biol Chem, 270:25805-25811, 1995; Thompson, L. D., et al., Biochem, 33:3831-3840, 1994). Therefore, there are likely other as yet undefined protein characteristics that must confer heparin-binding potential. Of relevance is the recent use of phage display technology to identify such novel heparin-binding sequences. This approach has generated three distinct HSPG-binding antibodies (van Kuppevelt, T. H., et al., J Biol Chem, 273 21:12960-12966, 1998). Significantly, one of the sequences (GRRLKD, SEQ. ID. NO:1) contained a heparin-binding consensus sequence, while the others (SLRMNGCGAHQ, SEQ. ID. NO:2, and YYHYKVN, SEQ. ID. NO:3) did not. The latter two lack significant basic charge, and thus may bind HSPGs through non-ionic interactions. All three anti-HS antibodies showed specificity for heparin and HS but not for other GAGs. Additionally, the antibodies all reacted differently towards HS from various sources, which would suggest a specificity in recognition of discrete HS molecules.
GAG structure may also play a role in determining binding affinity and selectivity for proteins. A classic example is the antithrombin-binding site on heparin, which is present on only about one third of heparin chains (Lam, L. H., et al., Biochem Biophys Res Commun, 69:570-577, 1976), but which has a thousand-fold greater affinity for antithrombin III than the overall heparin structure (Lee, M. K., and A. D. Lander, Proc. Nat Acad Sci USA, 88:2768-2772, 1991). Several other sequences or structural motifs have been identified in HS GAGs which underlie their binding interactions with basic fibroblast growth factor (bFGF) (Maccarana, M., et al., J Biol Chem, 268:23898-23905, 1993), lipoprotein lipase (Parthasarathy, N., et al., J Biol Chem, 269:22391-22396, 1994), and interleukin-8 (Lindahl, U., et al., J Biol Chem, 273:24979-24982, 1998). Other aspects of GAG fine structure also contribute to specific interactions with proteins. For example, for short basic peptides, heparin displays high affinities for sequences with contiguous clusters of basic amino acids, whereas HS displays high affinities for those sequences in which clusters of basic amino acids are separated by non-basic residues (Fromm, J. R., et al., Arch Biochem Biophys 343 (1):92-100, 1997). Such binding preferences may relate to the increased spacing between sulfates found throughout HS as compared with the more densely sulfated heparin. Heparin is capable of binding to a wide array of proteins, due to its high degree of flexibility and ability to “fit” itself into proteins.
Because of the presence of heparin-binding sequences in many physiologically important proteins, there was a need for small peptides with high affinities for heparin, or for heparin-like molecules (i.e., PGs, or other GAGs), to use in a variety of applications to modulate the activities of native GAGs and PGs. Therefore, in the present invention peptides with high affinities for heparin or for PGs have been designed to include heparin-binding consensus sequences; however, in doing so it was necessary to take into account previous studies showing that short peptides of native proteins do not behave like the native proteins, due to conformation and size limitations, and a lack of cooperativity in binding to various ligands. Thus, peptides were also designed including multiple consensus sequences arranged in tandem: (X-B-B-X-B-X)n or (X-B-B-B-X-X-B-X)m, where n=1-6, and m=1-5.
The basis for the design of the peptides of the present invention is the inclusion in their structure of multiple copies of sequences (including XBBXBX or XBBBXXBX, where X is a hydropathic amino acid and B is a basic amino acid), representing consensus sequences for heparin or PG-binding in natural proteins, and, in addition, may include the presence of a single cysteine residue preferably occupying, but not limited to, a position within a three residue distance of either the C- or N-peptide terminus, that promotes peptide dimer formation and greatly enhances peptide binding interactions with heparin. Any of these peptides may also be constructed of either L- or D-, or combinations of L- and D-amino acid isomer forms, or containing any amino acid in the X position of any peptide. Any of these peptides may also be used as carriers and/or integral components of various pharmaceuticals or bioactive agents targeted to interact with cell surfaces expressing PGs or heparin-like molecules, or to tissues which express PGs as cell surface or extracellular matrix components.
Current approaches to design peptides which bind to heparin include Wakefield et al. (U.S. Pat. No. 5,534,619, and U.S. Pat. No. 5,919,761; Wakefield, T. W., et al., J. Surg. Res., 56:586-593 1994; Wakefield, T. W., et al., J. Surg. Res., 63:280-286, 1996) and Harris et al. (U.S. Pat. No. 5,877,153). The Wakefield peptide sequences, specifically the grouping and spacing of the basic amino acids, are patterned after naturally-occurring protamines. The Harris et al. peptides are a series of single-chain and multi-chain peptides which incorporate arginines within a backbone of alanines. The spacings of the arginines are based on the heparin-binding sequence of antithrombin III. All the Harris peptides have AE as their N-terminal amino acid sequence.
The present invention, however, includes peptides based on the consensus sequences (XBBXBX) and (XBBBXXBX) determined by the analysis of a wide range of known heparin-binding proteins by Cardin et al (Cardin, A. D., and H. J. R. Weintraub, Arteriosclerosis, 9:21-32, 1989). The peptides designated in this application consist of as many as 6 repeating units of these sequences. These sequences are not found in protamine. In contrast to Wakefield et al., the peptides of the present invention contain repeating motifs with groups of two and 1 basic residues separated by a single alanine, or three and one basic residues separated by alanine-alanine. While single copies of these general sequences are associated with the heparin binding sites in many proteins, peptides derived from these proteins which include single copies of these sequences and their native surrounding amino acids have insignificant binding affinities for heparin. Furthermore, some proteins contain the Cardin type consensus sequences, but these sequences were shown not to bind heparin, and many other proteins bind heparin yet do not contain such consensus sequences. Thus it is not intuitive to use these types of sequences as heparin-binding agents.
Furthermore, the sequences used by Harris et al. mimic those found in a naturally occurring protein in terms of spacing and grouping of the basic residues, with no internal repeating structures, but the single-chain peptides have relatively weak ability to interact with heparin. Substantial binding is found only when multi-chain structures are formed. In contrast, the present invention involves, for the most part, single-chain peptides with repeating Cardin sequences. These peptides have a strong capability for binding to both unfractionated heparin and low molecular weight heparin.
A further difference between the peptides in the Wakefield and Harris patents resides in the engineering of alpha-helical structure into the peptides. Some of their peptides have partial alpha-helical structure. In the present invention, peptides are not alpha-helical in the native state, but assume an alpha-helical conformation when bound to heparin. Thus, the peptides of the present invention may have more flexibility to conform to a variety of heparin sequences encountered in any of the therapeutic heparin formulations.
An additional aspect of the present invention is the N-terminal-peptide sequences of the proteoglycan serglycin, which contain a single full or partial Cardin site near the N-terminus and a cysteine residue three amino acids from the C-terminus. These peptides dimerize through their cysteine residues and thus form a strong heparin-binding unit. Another feature of the present invention is the inclusion of cysteines near the C-termini of all the Cardin site peptides and the serglycin peptides to further enhance their heparin-binding functions.
The peptides of the present invention have a number of uses. One method of using these peptides is to promote cell attachment or adhesion to natural or synthetic surfaces.
Vascular diseases such as atherosclerosis, restenosis, and aortic aneurysms often result in permanent damage to blood vessels; typically, vessels become occluded as a result of vascular insult, causing decreased blood flow (Robbins, S. L. and R. S. Cotran, Pathological Basis of Disease. W. B. Saunders, Philadelphia. 598-613 pp., 1979). One approach to treatment of damaged vessels is surgical replacement of the diseased segment with an autologous or non-autologous native tissue graft (Zarge, J. I., et al., In Principles of Tissue Engineering, R. P. Lanza, et al., editors. Academic Press, Austin, 349-364, 1997). This approach is limited in that either healthy vessels must be removed (autologous) or a suitable donor vessel must be available (non-autologous). An alternative approach is the use of a synthetic vascular graft in place of a native tissue graft. In addition to not requiring an appropriate donor vessel or the removal and transplantation of a non-diseased vessel to the diseased area, synthetic vascular grafts can be modified to reduce complications from immune rejection or to increase patency rates and graft success (Munro, M. S., et al., Trans Am Soc Artif Intern Organs, 27:499-503, 1983; Leikweg, W. G., and L. J. Greenfield, Surg, 81:335-342, 1977; Park, K. D., et al., J Biomed Mater Res, 22:977-9227-29, 1988).
Historically, inert polymers composed of terephthalate (Dacron) or of expanded polytetrafluoroethylene (ePTFE) have been used to construct prosthetic vascular grafts (Zarge, J. I., H. P., and H. P. Greisler, In Principles of Tissue Engineering, R. P. Lanza, et al., editors. Academic Press, Austin, 349-364, 1997), but these materials typically invoke an immune response. Synthetic grafts can react with serum proteins and blood cells that can promote thrombus formation and lead to pseudointimal hyperplasia. (Zarge, J. I., H. P., and H. P. Greisler, In Principles of Tissue Engineering, R. P. Lanza, et al., editors, Academic Press, Austin, 349-364, 1997). Vascular replacement has been limited to large or medium size arteries where blood flow rates are high, outflow resistance is low, and as a consequence, the graft is less likely to become occluded by a thrombus. Conversely, small arteries are more prone to graft failure via thrombosis or hyperplasia because of lower flow rates and higher outflow resistance. An inappropriate infiltration of smooth muscle cells during the healing process can also result in vessel occlusion. Control of this immune response and smooth muscle cell infiltration could occur in a vessel lined with endothelial cells, which secrete factors inhibiting platelet and erythrocyte aggregation (Fantone, J. C., and P. A. Ward, In Pathology, E. Rubin, and J. L. Farber, editors. J. B. Lippincott Co., Phil. 43, 1994), as well as factors that inhibit smooth muscle cell proliferation, but, endothelial cells typically fail to proliferate well on these graft materials. (Zarge, J. I., H. P., and H. P. Greisler, In Principles of Tissue Engineering, R. P. Lanza, et al., editors, Academic Press, Austin, 349-364, 1997). Attempts have been made to overcome these limitations by coating the graft with anticoagulants to limit thrombus formation, growth factors to promote endothelial cell proliferation, or proteins with antiproliferative effects on smooth muscle cells. The presence of endothelial cells in the transplanted graft, however, is thought to increase the chance of survival of the graft (Herring, M. B., et al., Surgery. 84:498, 1978). Studies in which prosthetic vascular surfaces were seeded with autologous endothelial cells before transplantation displayed an increase of 30% in patency rates over three years in comparison to non-seeded surfaces (Zilla, P., et al., J Vasc Surg, 19:540-548, 1994). The obvious limitation of pre-seeding, however, is the need to harvest and culture endothelial cells to the appropriate density prior to seeding, as well as generating vascular graft materials with surface properties optimized for endothelial cell attachment and proliferation.
Endothelial cells carry a negative surface charge (Vargas, F. F., et al., Membrane Biochemistry. 9:83, 1990) that can inhibit platelet adherence, and they express a variety of GAGs on their surface that bind the anti-coagulant anti-thrombin III (Mertens, G., et al., J Biol Chem, 267 (28):20435-20443, 1992). Vargas and co-workers have shown that sulfated GAGs are the main carriers of surface charge on vascular endothelial cells, primarily as heparan sulfate (HS) and chondroitin sulfate PGs (Vargas, F. F., et al., Membrane Biochemistry. 9:83, 1990). Specific types of PGs on endothelial cell surfaces include the syndecans and glypican (Mertens, G., et al., J Biol Chem, 267 (28):20435-20443, 1992).
Thus, the surface chemistry (i.e., the predominance of its PG component) of endothelial cells will prove useful as a means of tethering and maintaining these cells in a transplanted synthetic vascular graft. One goal of the present invention is to discover peptides with high affinities for endothelial cell surface PGs. Such peptides are used by covalently attaching them to synthetic vascular grafts, and in the presence of endothelial cells, promote their attachment to the graft surface, thereby increasing the probability of graft success.
Another use of the peptides of the present invention is for heparin-and PG-binding as modulators of hemostasis via interactions with endothelial cells and as anti-heparin therapy in plasma. These peptides of the present invention function as agents for neutralization of unfractionated heparin, low molecular weight heparin, or Orgaran (Organon, mixture of chondroitin sulfate/heparan sulfate/dermatan sulfate) overdose.
Currently, the only FDA-approved heparin antidote available is Protamine. Protamine can cause several serious side effects in patients, and although Protamine is effective in humans against unfractionated heparin, it is not effective against low molecular weight heparins or against Orgaran. Since Protamine is a natural product that is an undefined mixture of amino acids, its content is variable across different preparations, and thus dosage is uncertain, presenting problems in its clinical use.
The peptides of the present invention are useful for counteracting the actions of heparin and other anticoagulant glycosaminoglycans on thrombin and Factor Xa activity, and may affect other proteins as well. Heparin is used routinely for anticoagulation. The interactions of exogenously administered heparin with the proteins of the coagulation and fibrinolytic pathways have been summarized in detail (Conrad, H. E., Heparin-Binding Proteins, Academic Press, San Diego, 1998). These interactions are complex on many levels. The best-characterized targets for heparin are the procoagulant proteins thrombin and Factor Xa, which are inhibited by AT III when heparin binds to AT III. However, heparin acts at many sites. In some cases, the effect of heparin is anticoagulant and in other cases procoagulant. Some proteins, e.g. AT III, have heparin-binding consensus sites. However, the putative heparin-binding sequences are different for every known protein in these pathways, and the effects may depend on the 3-dimensional relationships of basic residues resulting from protein folding, rather than a short linear sequence, as is known for the binding of heparin to AT III (Carrell, R. W., et al., Structure 2:257-270, 1994). A tetrameric protein conformation of platelet factor 4 (PF4) is required for long-chain heparin binding (Rucinski, B., et al, Thromb Hemostas 63:493-498, 1990; Ibel, K., et al, Biochim Biophys Acta 870:58-63, 1986; Talpas, C. J., et al, Biochim Biophys Acta 1078:208-218, 1991). Formation of a two-protein complex (PAI-1/vitronectin) involves the vitronectin heparin binding site (Kost, C. W. et al, J Biol Chem 267:12098-12105, 1992; Deng, G., et al, J Cell Biol 134:1563-1571, 1996) and therefore could be disrupted by heparin. The inactivated AT III/thrombin complex is released from the endothelial surface, binds as a complex to vitronectin, and then is taken up for catabolism by binding of the vitronectin heparin-binding domain to HSPG on the endothelium (Hogasen, J., et al, J Biol Chem 267:23076-23082, 1996; deBoer, H., et al, J Biol Chem 94:1279-1283, 1993).
Heparin is a complex mixture of polysaccharides. Some of the interactions require long-chain heparins (AT III for inactivation of thrombin and binding to thrombin, HC II, PF4, and thrombospondin) while others depend on or can function with low molecular weight heparin chains (AT III for inhibition of Factor Xa, vitronectin, TFPI) (Conrad, H. E., Heparin-Binding Proteins, Academic Press, San Diego, 1998). To further complicate the situation, specific sequences within the heparin chains may be required for interactions with the different proteins (Conrad, H. E., Heparin-Binding Proteins, Academic Press, San Diego, 1998), and all naturally-occurring heparins and heparan sulfates are very diverse in their carbohydrate structures. The catabolism of the higher molecular weight heparins in the plasma results in a constantly changing spectrum of actual heparin chains that are available for reaction with the various proteins, and thus the nature of the possible anticoagulation or fibrinolytic reactions will change over the hours after the dosage is given. Finally, many other plasma proteins that are not involved in the coagulation or fibrinolytic processes can bind heparin, and variations in the concentration and nature of these proteins in different individuals can influence the availability of heparin for these two pathways. Thus specific single peptides or combinations of peptides may target specific interactions between heparins and cell surface or plasma proteins to get the greatest effectiveness and minimize adverse reactions
It is often necessary to reverse the effects of heparin when anticoagulation has reached a stage at which hemorrhage becomes a threat, notably after the routine use of heparin for anticoagulation during cardiopulmonary bypass, and in patients who develop an endogenous heparin-like coagulation inhibitor. The most commonly used anti-heparin drug is protamine, a mixture of basic proteins from fish sperm nuclei, that contains a high concentration of the amino acid arginine. When injected into a person who has been treated with heparin, it complexes rapidly to the heparin, thereby neutralizing its activity. Protamine also has numerous side effects including pulmonary hypotension that are difficult to control and provide significant health risks to the patient. Also, since Protamine is a poorly-defined and potentially variable product, dosage determination can be problematic. Importantly, Protamine has been shown to be ineffective for neutralization of low molecular weight heparins and the non-heparin glycosaminoglycan anticoagulant Orgaran. Well-defined heparin-or other GAG-binding peptides could be of considerable utility for reversing overdose of these specific anticoagulant preparations. Carson and co-workers (Lui, S., et al, J Biol Chem 94:1739-1744, 1997) have identified a heparin-binding peptide from an epithelial/endothelial cell surface protein that has some ability to neutralize heparin effects on thrombin generation, but optimal effects were found only at high peptide concentrations and low heparin and low thrombin concentrations. Preliminary data in the present invention suggest that the Cardin and serglycin peptides reverse the heparin effect on thrombin at several-fold lower peptide concentrations and 7-fold higher thrombin concentrations than the peptide described by Carson and co-workers. We have also shown that several of the peptides are effective neutralizers of low molecular weight heparin (Enoxaparin, Lovenox) and Orgaran in vitro, and of Lovenox in vivo in rats, in accordance with their affinity constants for low molecular weight heparin in vitro. Thus the peptides described in this application may have important clinical applications, especially if they can be targeted to specific reactions in the relevant pathway and to specific classes of heparins.
Another use for the peptides of the present invention is to block the uptake and clearance of heparin by blocking uptake receptors on tissue, without binding to the circulating heparin itself, and thus prolonging the half-life in the circulation. Such an agent would reduce the frequency of administration of the drug, as well as the amount needed. This could be especially useful for home-based therapy with low molecular weight heparin, which is administered by subcutaneous injection and is becoming the standard for post-hospitalization anticoagulation.
Multiple interactions between the proteins of the coagulation and fibrinolysis pathways and endothelial cell surface PGs generate a complex surface on which ongoing coagulation and fibrinolysis are normally balanced to create a non-thrombotic state. The heparan sulfate PGs (HSPGs) of the endothelium mediate antithrombotic/anticoagulant function through binding and activation of Antithrombin III (AT III) and binding of tissue factor pathway inhibitor (TFPI). AT III bound to endothelium heparan sulfate can inactivate both thrombin and Factor Xa. TFPI binds to Factor Xa and this complex then interacts with the Factor VIIa/tissue factor complex to inactivate both Factors VIIa and Xa. Adherence of TFPI to the endothelium via the HSPG protects against proteolysis of the heparin-binding C-terminal domain (Nordfang, O., et al, Biochem. 30:10371-10376, 1991); without this domain, activity is lost. Heparin-binding peptides such as those described in this study could behave similarly to platelet factor 4 (PF4) in that they could bind to the heparan sulfates on the endothelial surface. For example, docking of a peptide onto the heparan sulfate chain in a reversible manner could protect the GAG from degradation by platelet heparitinase released by aggregating platelets at the site of a developing thrombus, leaving the GAG able to resume its antithrombotic function in a shorter time frame than would be required for resynthesis. On the other hand, a peptide with a very high affinity for the AT II-binding sequences of endothelial heparan sulfate could block the binding and therefore the activity of AT III and provide a more favorable surface for clot formation, thus promoting wound healing.
Therefore, in some embodiments, the peptides of the present invention have affinity for heparin/heparan sulfate on cell surfaces and can be used as agents to promote healing, either by injection or by topical application. Injection or topical application of the peptides alone also might serve to assist wound-healing by dislodging ATIII and/or tissue factor pathway inhibitor (TFPI) from their binding sites and subsequently blocking these binding sites on the endothelium of broken blood vessels, thereby reducing the anticoagulant activity of the surface and enabling a clot to form. Alternatively, contemporaneous injection or application of a mixture of heparin and a heparin-binding peptide could generate a molecular complex, or low affinity heparin sink, that will then transfer the heparin to proteins with greater heparin-binding affinities.
The peptides of the present invention can be used to bind and neutralize or activate, or otherwise modulate the actions of various PGs or GAGs, thereby influencing their growth- or differentiation-modulating activities. For example, heparin and heparin-like molecules such as cell surface HSPGs are known to inhibit smooth muscle cell proliferation, to potentiate the activities of growth factors like basic or acidic fibroblast growth factor on endothelial cells, and to inhibit or promote cell differentiation of smooth muscle cells, chondrocytes, and other cell types. The peptides described here could be used to modulate the actions of heparin or endogenous heparan sulfate PGs, with significant consequences to cell growth and differentiation.
GAGs exhibit a wide variety of potent activities on cell growth, migration, differentiation, metabolism, and adhesion (Jackson, R. L., et al., Physiol Rev, 71:481-539, 1991; San Antonio, J. D., and R. V. Iozzo, Encylc Life Sci, In Press, 1999). One of the earliest reports of an effect of GAGs on cell growth reported that fibroblastic mouse L cells in suspension culture exposed to 50 μg/ml heparin were growth inhibited by seventy-three percent (Karnovsky, M. J., et al., Annals of the New York Academy of Science, 556:268-281, 1989). Several strong antiproliferative activities of GAGs on a variety of cell types have been reported since (San Antonio, et al., Connective Tissue Res, 37:87-103, 1998). The effects of heparin on vascular smooth muscle cells (VSMC) have been the most extensively studied owing to the relevance of this topic to vascular disease. Although for some cell types heparin antiproliferative action may involve displacement of HS or heparin-binding growth factors from cell surface receptors, for VSMC heparin may also be internalized and act directly in the cytoplasm and nucleus (Karnovsky, M. J., et al., Annals of the New York Academy of Science, 556:268-281, 1989). An important component of vascular diseases including atherosclerosis and restenosis is the pathological growth of vascular smooth muscle cells. As GAGs are strong regulators of VSMC growth they are potentially useful in treating these diseases. The effect of heparin on VSMC growth in vivo was first discovered in experiments aimed at determining whether heparin may inhibit the response to injury cascade of accelerated atherosclerosis owing to its antithrombotic activity; a dramatic inhibition of VSMC proliferation by heparin was observed (Karnovsky, M. J., et al., Annals of the New York Academy of Science, 556:268-281, 1989). It was next shown that the growth effect of heparin on VSMC in vivo is exhibited by either anticoagulant or non-anticoagulant fractions, and that these effects are mimicked by heparin or HS on VSMC in vitro (Karnovsky, M. J., et al., Annals of the New York Academy of Science, 556:268-281, 1989). It has been proposed that in the healthy vascular wall, endothelial-derived HS maintains VSMC in a quiescent growth state, but that injuries which result in endothelial denudation remove this paracrine mechanism, resulting in uncontrolled VSMC proliferation and vascular lesion formation (Karnovsky, M. J., et al., Annals of the New York Academy of Science, 556:268-281, 1989). Thus, the peptides described here are useful in neutralizing the antiproliferative activities of endogenous or exogenous heparins or heparan sulfates on vascular smooth muscle cells or other cell types. For example, the peptides may be used to neutralize endothelial cell-derived HSPG's during vascular wound healing.
Heparins and heparan sulfates have been shown to promote cartilage development at low concentrations, and to inhibit it at high concentrations (San Antonio, J. D., et al., Devel Biol, 123:17-24, 1987). Thus, the peptides described here may prove useful as modulators of cartilage differentiation, especially in instances where cartilage tissue scaffolds are being constructed for autologous tissue transplants, e.g., for use in orthopedic surgical applications.
Tumor matrix stromas may play important roles in potentiating tumor growth and metastasis (Iozzo, R. V., Lab Invest, 73:157-160, 1995). For example, increases in perlecan expression are seen during development of colon carcinomas and of malignant melanomas; its HS chains may potentiate growth factor activity and induce angiogenesis surrounding the tumor, thereby enhancing its growth (Nugent, M., and R. V. Iozzo, Internat J Biochem. and Cell Biol, In Press, 1999). Furthermore, the binding selectivity of HS chains for various members of the fibroblast growth factor family can be influenced by fine structural features such as the patterns of 6-O-sulfation and the abundance of sulfated domains (Lindahl, U., et al., J Biol Chem, 273:24979-24982, 1998). A pathological role of tumor cell surface PGs has also been suggested. For example, Chinese hamster ovary cells carrying various mutations of PG synthesis were injected into nude mice and tested for their tumorigenic abilities. Mutants which expressed low levels of PGs failed to produce tumors, and of those with normal PG levels but with defects in the synthesis of specific GAG types, the structure of HS, but not of CS, was most important to their tumorigenicity (Esko, J. D., et al., Science, 241:1092-1096, 1988). Thus, if the peptides described here exhibit GAG type specific or GAG sequence specific binding preferences, they may be useful in directly modulating the function of tumor cell GAGs or PGs, or as carriers of drugs to be targeted to control the growth of, or to kill tumor cells expressing unique GAGs or PG variants.
PGs secreted by normal cells are proposed to play a key barrier function by inhibiting the migration of tumor cells across basement membranes. However, tumor cells have been shown to secrete the enzyme heparatinase, which degrades the HS chains within basement membranes, thereby potentially enabling such malignant cells to breach the basement membrane, enter the circulation, and spread throughout the body (Katz, B. Z., et al., Invasion and Metastasis, 14:276-289, 1994-5). The peptides described here could thus be used as inhibitors of GAG hydrolase-mediated tumor metastasis.
Another key component of tumor growth and survival is proposed to be the development of a blood vessel supply to the tumor (Folkman, J., and M. Klagsburn, Science, 235:442-447, 1987). Tumor angiogenesis is strongly inhibited by the heparin-binding protein endostatin (O'Reilly, M. S., et al., Cell. 88 (7):277-285, 1997), and in vitro, heparin is required to promote angiogenesis in response to growth factors (Jackson, C. J., et al., Exp Cell Res, 215:294-302, 1994). The peptides described here could therefore function as inhibitors of growth-factor dependent angiogenesis in vivo, therefore inhibiting tumor growth.
Yet another application of the present invention is the targeting of drugs to cell surfaces of endothelium or other cell types which express PGs. For example, drugs to be targeted to endothelial cells could be complexed with the peptides described here, or the peptide sequences could be integrated into the drug, and then the drug could be administered to the systemic circulation. The peptide component of the drug would mediate high affinity interactions with the endothelial cell surface, effectively delivering the drug for action at that site, or potentially promoting the cellular uptake of the drug.
Since endothelial cell surface charge is largely due to cell surface GAGs and PGs (Vargas, F. F., et al., Membrane Biochemistry, 9:8, 1990), and the peptides described in this patent exhibit high affinity interactions with endothelial cell PGs, then the peptides can be applied as tools to deliver drugs to endothelial cells in vivo. For example, a drug which is designed to act on endothelial cells could be complexed with the peptides either covalently or non-covalently, and delivered to the systemic circulation. The peptide component of the complex would facilitate high affinity interactions with the endothelial cell surface, thereby bringing the drug in contact with the endothelial cell surface to exert its activity there, or to facilitate its uptake by the endothelial cells. Such a use for these peptides is not limited to endothelial cells, since many cell types in the body express distinct classes or types of PGs. Furthermore, within each type of cell population, structural variants of GAGs and PGs may be expressed, thereby distinguishing these cell variants on a structural and functional level. Thus, for example, it has been shown that normal B cells and various transformed (cancerous) B cells express different variants of syndecan-1 which show distinct differences in the chemistry of their heparan sulfate chains (Sanderson, R. D., et al., J Biol Chem, 269:13100-13106, 1994). If some of the peptides described here show binding preferences for the heparan sulfates expressed on the cancerous B cells, then such peptides could be used as carriers of drugs targeted to those cells. Finally, another potential target cell for the peptides described here are the chondrocytes, which are present in all joint surfaces and which express high amounts of sulfated GAGs in their pericellular spaces. Previous work has shown that the basic endogenous protein lysozyme accumulates in joints, likely owing to its interactions with cartilage GAGs (Keuttner, K., et al., Clin Orthop Relat Res, 112:316-339, 1975). These results suggest that basic peptides such as those described here, when injected systemically or directly into or near joints, should also concentrate and/or be retained in cartilagenous regions. Therefore, systemic application of the peptides described here, complexed with drugs targeted to chondrocytes for treatment of, for example, arthritic diseases, could potentially find many uses.
Another application of the peptides of the present invention is their use to modulate the activities of enzymes that act on GAG substrates. For example, GAG hydrolases including some of the heparinases and heparatinases contain heparin-binding consensus sequences which they are proposed to use in binding to their GAG substrates. The peptides described here could be used to inhibit this binding through competition, thereby inhibiting the activity of the enzymes.
Several of the enzymes that hydrolyze heparin and heparan sulfates are used commonly in scientific investigations to characterize the structure and function of GAGs within tissue and cell preparations. Furthermore, these enzymes are important natural products as they are secreted by specific types of bacteria, are present in the venom of some poisonous snakes, and are secreted by normal human cells, and by human tumor cells, where they are proposed to promote tumor cell metastasis (Katz, B. Z., et al., Invasion and Metastasis, 14:276-289, 1994-5; Sasisekharan, R., et al., Proc Natl Acad Sci USA, 90:3660-3664, 1993). Since these enzymes contain heparin-binding consensus sequences that are proposed to mediate the interactions between the enzyme and their substrates, the peptides described here will serve as effective inhibitors of enzyme action for many in vitro and in vivo applications.
Yet another use of the peptides of the present invention is in the affinity purification of bioactive sequences of GAGs. For example, some heparin-binding proteins have been shown to interact with specific sequences or domain structural features on heparins or heparan sulfates, including antithrombin III, lipoprotein lipase, and laminin. Thus, the peptides described here may similarly exhibit binding preferences for distinct sequences in GAGs, making them useful as affinity matrices for the purification of specific GAG sequences for a variety of uses.
Heparin-binding proteins have been shown to interact with specific sequences or domain structural features on heparins or heparan sulfates, including ATIII (Lam, L. H., et al., Biochem Biophys Res Commun, 69:570-577, 1976), lipoprotein lipase (Parthasarathy, N., et al., J Biol Chem, 269:22391-22396, 1994), and laminin. For example, the determinant on heparin necessary for AT-Ill binding was located on only about one third of heparin chains, and is a pentasaccharide sequence composed of a 6-O-sulfated glucosamine in the first position, a 3-O-sulfated central glucosamine, two N-sulfated glucosamines, and a carboxylated iduronic acid (Jackson, R. L., et al., Physiol Rev. 71:481-539, 1991). To purify this sequence from heparin is an expensive endeavor as it requires heparin fragmentation followed by affinity chromatography on ATIII columns. However, if any of the peptides described here showed binding preference for specific sequences such as the ATIII binding site, then they could be used as low cost affinity matrices for the large scale purification of bioactive GAG sequences and fragments. Furthermore, such approaches could potentially be useful in endeavors to sequence or functionally characterize GAG samples of unknown chemistries, if libraries of heparin-binding peptides contain peptides with unique binding selectivities for distinct features of heparin or heparan sulfate chemistry, these could be used as tools fractionate, isolate, and quantitate specific GAG sequences from complex GAG mixtures.